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Abstract

Introduction

The hypothalamic-pituitary-adrenal axis is known to be related to abdominal symptoms,
and the relationship between abdominal pain and cortisol secretory patterns has been
previously investigated using a cross-sectional approach. Here, we investigated the
effect of day-to-day variations in salivary cortisol and dehydroepiandrosterone-sulfate
levels on abdominal symptoms in healthy individuals.

Methods

Eleven college students (4 males and 7 females) participated in this study. The participants
were asked to collect their saliva immediately after awakening and before bedtime
for eight consecutive days. They also completed a questionnaire about abdominal symptoms
before bedtime. The linear mixed model was applied to analyze the effects of the day-by-day
variability or the 8-day average adrenal hormone level (at awakening, before bedtime,
slope from awakening to bedtime) on abdominal symptoms.

Results

The day-to-day variability of cortisol levels before bedtime was negatively related
with loose stool, while the day-to-day variability of the cortisol slope was positively
correlated with loose stool. A low 8-day average dehydroepiandrosterone-sulfate level
at awakening was positively related with frequent bowel movements, loose stool, and
long bouts of severe abdominal pain. Likewise, a low 8-day average dehydroepiandrosterone-sulfate
slope was positively related with long bouts of abdominal pain.

Conclusions

Low cortisol levels before bedtime and a steeper diurnal cortisol slope during the
day may be related to bouts of diarrhea during the day.

Introduction

The effect of changes in the hypothalamic-pituitary-adrenal (HPA) axis on abdominal
symptoms has been previously reported. On the basis of the results of previous studies,
Mayer et al. (2002) suggested that complex interrelationships exist between gut-associated
immune tissue, the central nervous system, and the enteric nervous system [1]. Psychosocial stressors activate stress circuits within the emotional motor system,
and the resulting peripheral output is manifested in the form of cortisol, corticotrophin-releasing
factor, and autonomic (norepinephrine and epinephrine) responses, which induces the
mucosal immune system to activate a Th2 response [2,3]. An increase in mast cells brought about by alterations in the Th1/Th2 balance may
stimulate bowel movements by altering the access of luminal organisms and antigens
to the gut immune system [4,5].

Previous studies have suggested a relationship between abdominal pain and the cortisol
response levels in the morning and evening. Patients with functional gastrointestinal
disorder have been shown to have lower cortisol levels 60 min after awakening as compared
with healthy controls although there was no significant difference in the cortisol
response after awakening between these groups. Furthermore, total secretion (area
under the curve) of cortisol during the first hour after awakening was also found
to be lower in patients with functional gastrointestinal disorder than healthy controls
[6]. Compared with controls, patients with irritable bowel syndrome (IBS) showed significantly
higher cortisol levels in the morning and lower levels in the evening while maintaining
the physiological circadian fluctuation (i.e., morning cortisol levels higher than
in the evening) [7]. Moreover, a previous study suggested that in children with recurrent abdominal pain
of psychosomatic origin, levels of cortisol in the morning and total secretion were
significantly higher than those in healthy children [8]. Another study reported that children with recurrent abdominal pain showed higher
cortisol levels in the morning than healthy children [9]. Although these studies do not present consistent findings, it is likely that there
exists a relationship between cortisol secretion and abdominal symptoms. Indeed, the
previous studies clearly show that there is a difference in cortisol secretion between
groups with and without functional gastrointestinal symptoms.

It has been reported that variations in the HPA axis marker have an impact on abdominal
symptoms, and the relationship between abdominal pain and cortisol secretory patterns
has been investigated using the cross-sectional approach. However, the following problems
must be addressed. First, in addition to investigating the effect of cortisol secretory
patterns on abdominal symptoms, we must examine day-to-day intraindividual variations
in cortisol secretion in relation to the severity of abdominal symptoms. Second, it
is important to clearly elucidate the relationship between abdominal symptoms and
dehydroepiandrosterone (DHEA). DHEA is the most important androgen secreted by the
adrenal cortex and is a precursor for sex steroids; the main secretagogue for DHEA
is thought to be adrenocorticotrophic hormone. DHEA is thought to affect immunological
function in a manner opposite to that of cortisol [10]. DHEA is converted into DHEA-sulfate (DHEA-S) and accumulated within a relatively
short time after secretion. Therefore, DHEA-S is the major circulating form, and is
converted to DHEA by a sulfotransferase in the tissue. In this study, we collected
saliva after relatively long intervals to determine DHEA-S, which has an apulsatile
secretion pattern and which was more useful for our study purposes than DHEA, which
has a pulsatile secretion pattern. Thus, analysis of both cortisol and DHEA-S may
be useful with regard to studying the effects of adrenal hormones on abdominal symptoms.

We aimed to examine the relationship between physiological factors and abdominal symptoms
by assessing day-to-day intraindividual variations in cortisol and DHEA-S secretion
patterns. For this, we investigated the effect of daily changes in adrenal hormone
(salivary cortisol and DHEA-S) levels on abdominal symptoms by monitoring the subjects
for eight days.

Methods

Participants

The participants were 11 college students (4 males and 7 females, mean age = 26.9
± 2.8 yrs). Mean body mass index (BMI) of the participants was 20.9 kg/m2. No one used any habitual medications or dietary supplements that affect HPA activity
or had any history of gastrointestinal surgery for the past five years. Written informed
consent was obtained, and the study was approved by the university's ethics committee.

Measures (diaristic questionnaire)

1) Information related to abdominal symptoms

The following items were assessed: "Bowel movement," "Stool properties," "Duration
of abdominal pain (minutes)," and "Degree of abdominal pain."

"Bowel movement" was assessed by asking the following question: "How many bowel movements
did you have today?;" "Stool property" was based on a rating of 1 to 8 (1: no bowel
movement; 2: lumpy stool; 3: hard stool; 4: like a banana with cracks on its surface;
5: like a banana, smooth and soft; 6: soft blobs with clear-cut edges; 7: fluffy pieces
with ragged edges, mushy; 8: watery, no solid pieces); "Duration of abdominal pain"
was assessed by asking the following question: "How long did you experience continuous
abdominal pain today?;" and "Degree of abdominal pain" was rated from 0 (not at all)
to 10 (very severe).

2) Information about sleep duration

Sleep duration was calculated from the time subjects went to sleep to the time they
awakened. Previous research has noted a correlation between cortisol secretion levels
and sleep duration [11].

3) Perceived Stress Scale

The Perceived Stress Scale 4 (PSS4) [12,13] was designed to measure the degree to which situations in one's life are appraised
as stressful. The PSS4 includes four items, each of which are rated from 0 to 4. In
this study, the PSS4 was used in order to consider the effect of psychological stress
on adrenal hormone secretion.

Determination of adrenal hormone levels

Participants were asked to collect their saliva samples at awakening and immediately
before bedtime for eight consecutive days. The samples were used to determine cortisol
and DHEA-S levels. Saliva samples used to determine cortisol levels were collected
using Salivette swabs (Sarstedt Ltd., Germany.). For samples for determining DHEA-S
levels patients were asked to expectorate saliva through a short plastic straw into
a collection vial. Saliva samples were stored at room temperature (10-20°C) and centrifuged
at 3000 rpm for 5 min within three days after sampling. The concentrations of cortisol
and DHEA-S in the saliva were determined by an enzyme immunoassay using an EIA Kit
(Cortisol: Salimetrics LLC, USA; DHEA-S: Diagnostic Systems Laboratories, Inc., USA).

Procedure

Participants were asked to collect their saliva on awakening and immediately before
bedtime. They were also asked to complete the diaristic questionnaire before bedtime
for eight consecutive days. The participants were asked to not drink alcohol during
the experimental period. Further, we avoided selecting female subjects who were menstruating
during the period so that menstrual pain would not be confused with bowel pains.

Statistical Analysis

For the analysis of adrenal hormone data, cortisol and DHEA-S concentration values
were square root transformed. The relationship between adrenal hormone, abdominal
symptoms, stress, and sleep duration was analyzed using Pearson's product-moment correlation
coefficient. Student's t test for repeated measures was applied to analyze the cortisol and DHEA-S slopes between
awakening and bedtime. The linear mixed model was applied to analyze the effect of
adrenal hormones on abdominal symptoms. The abdominal symptoms were dependent variables
and the 8-day average adrenal hormone level, the daily intraindividual variability
of adrenal hormones (at awakening, before bedtime, slope from awakening to bedtime
[the value at awakening minus the value before bedtime]), the PSS4 score, and the
sleep duration were covariates. Data analysis was performed using the SPSS 15.0 software
(SPSS Japan Inc.).

Effect of adrenal hormone slopes from awakening to bedtime on abdominal symptoms

The results of the linear mixed model with abdominal symptoms as dependent variables
and the day-to-day intraindividual variability, the 8-day average adrenal hormone
slopes, the PSS4 score, and sleep duration as covariates are shown in Table 5. The day-to-day variability of the cortisol slope was positively related to stool
property (t = 2.07, p < 0.05), while the 8-day average DHEA-S slope was negatively related to the duration
of abdominal pain (t = -2.07, p < 0.05).

Discussion

This is the first study to investigate the effect of the day-to-day variability of
adrenal hormone levels on abdominal symptoms in healthy individuals. There is a possibility
that a steeper diurnal cortisol slope during the day and lower cortisol levels in
the evening is related to bouts of diarrhea. The results of this study are similar
to those of two previous studies [7,14]. Previous research suggests that compared with control patients, IBS patients show
significantly higher levels of cortisol in the morning and lower levels in the evening
[7]. Therefore, the cortisol secretion patterns influencing vulnerability to abdominal
symptoms may be similar in healthy individuals and individuals with IBS. Moreover,
Sugaya et al. [14] reported that individuals with IBS had lower cortisol responses under acute stress.
The result of this study is similar to that of Sugaya et al. [14] with respect to the relationship between the lower cortisol response and abdominal
symptoms. The results of the present study are similar to those of Patachioli et al.
[7] and Sugaya et al. [14]; however, the analysis of the day-to-day variability of cortisol provided a more
definitive conclusion. The previous studies only indicated the characteristic of cortisol
at a specific point in time. From these results, we indicate the continuity between
healthy individuals and individuals with IBS with respect to the relationship between
cortisol and abdominal symptoms.

We found no significant effects of day-to-day variability of DHEA-S on bowel symptoms.
However, the 8-day average DHEA-S level at awakening was negatively related to all
variables of abdominal symptoms. These results suggest that chronically reduced levels
of DHEA-S at awakening or baseline levels are related to the degree and duration of
abdominal pain, diarrhea, and infrequent bowel movements. In addition, the 8-day average
of the DHEA-S slope was related to the duration of abdominal pain. Therefore, a flat
circadian rhythm of DHEA-S can increase the duration or frequency of abdominal pain.
Moreover, the 8-day average DHEA-S level before bedtime was negatively related to
the frequency of bowel movements and stool property. In fact, we assert that low DHEA-S
levels may have an affect on the frequency of bowel movements and stool property to
a higher degree than the slope between the morning and the evening.

Previous research has suggested the possibility that higher cortisol and lower DHEA
(or DHEA-S) levels may result in altered immune function, which in turn may cause
abdominal pain [2,5,10]. However, our findings do not suggest that higher cortisol levels affect abdominal
symptoms. Sugaya et al. [14] indicated that the secretion of a secretagogue, which induces the production of an
adrenal hormone (e.g., adrenocorticotrophic hormone and corticotrophine-releasing
hormone), may be normal in individuals with IBS because they showed a lower cortisol
response than healthy controls and their DHEA profiles were similar to those of healthy
controls. Therefore, considering the results of the present study and those of Sugaya
et al. [14], there is a possibility that lower cortisol levels may aggravate abdominal symptoms
due to a dysfunction of negative feedback regulation to corticoprophin-releasing hormone
secretion. We can speculate that chronically low DHEA-S levels may cause the deterioration
of abdominal pain and diarrhea with respect to a decreased effect of DHEA-S on immunological
function that is opposite to cortisol.

This study provides some new insights and provides valuable information for future
research; nevertheless, there were three limitations to the present work. First, we
were unable to consider the relation between the participants dietary habits and their
abdominal symptoms. Although inclusion of items about dietary habits may yield more
accurate results, we feel that future studies should employ a less demanding saliva
sampling process over the 8-day period. Second, the diurnal cortisol slope was calculated
by only determining cortisol levels immediately after awakening and before bedtime;
samples were not obtained for any other times. Hence, our results may not be useful
for determining the cause of abdominal pain taking place early in the day. In the
future, the relationship between the circadian rhythm of adrenal hormones and abdominal
symptoms must be determined by taking multiple saliva samples throughout the day.
Therefore, future studies should aim to maintain a good balance between consideration
for the participants and collecting multiple samples. Third, the number of participants
in this study was rather small.

Conclusions

This is the first study to investigate the effects of day-to-day variations in salivary
cortisol and DHEA-S secretion on abdominal symptoms. The results of our study suggest
that variations in adrenal hormone levels affect the duration and severity of abdominal
symptoms. Day-to-day variations in cortisol levels before bedtime and a steeper cortisol
slope may be positively related to a tendency toward bouts of diarrhea. On the other
hand, a persistent lower baseline and a flatter slope with regard to DHEA-S may also
be negatively related to abdominal symptoms. The results of our study suggested the
possibility that in the future the accurate clinical condition of abdominal symptoms
may be able to be revealed by a device that can take multiple saliva samples throughout
the day and by better controlling various daily habits.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

NS conceptualized and designed the study, collected the data, conducted the saliva
analysis, performed the statistical analysis, and drafted the manuscript. SI, NO,
KS, HK, KY, and HT conceptualized and designed the study, collected the data, and
conducted the saliva analysis. SN and HS conceptualized and designed the study. All
authors read and approved the final manuscript.

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